2.1. Passive Immunotherapy
Passive immunotherapy (also termed passive immunization) refers to the administration of an immunoreactive reagent (i.e., an antibody) comprising, for example, an antigen binding region directed against an epitope on a pathogen, tumor or pathogenic protein, and an Fc receptor-binding region, directly to a patient. The immunoreactive reagent can be given prophylactically to, for example, inhibit infection, or therapeutically to reduce or eliminate infection, to reduce or eliminate cancer cells, or to clear or remove pathogenic proteins, e.g., protein aggregates or deposits, as occurs in neurodegenerative and/or amyloidogenic disease. This is distinguished from immunization of a patient with a protein to induce an in vivo immune response to produce antibodies. Such administration preferably results in the stimulation of effector cells with Fc receptors capable of interacting with the Fc portion (i.e., the Fc receptor binding region) of the antibody or immunoreactive agent, resulting in cellular immune functions such as antibody-dependent cellular cytotoxicity (e.g., ADCC) or antibody-mediated opsonization and/or phagocytosis directed against the cell, pathogen, or protein possessing the epitope recognized by the immunoreactive agent. The saponin-mediated enhancement of passive immunotherapy can occur through stimulation of effector cells, i.e., induction and/or activiation of the Fc receptors on such cells. The efficacy of antibody-mediated tumor therapy which depends on FcR effector cell functions can be modified by the use of specific cytokines. Keler, et al., 2000, J. Immunol. 164:5746-5752.
2.2. Saponins
Quillaja saponins are a mixture of triterpene glycosides extracted from the bark of the tree Quillaja saponaria. They have long been recognized as immune stimulators that can be used as vaccine adjuvants, (Campbell and Peerbaye, 1992, Res. Immunol. 143(5):526-530), and a number of commercially available complex saponin extracts have been utilized as adjuvants. Crude saponins have been extensively employed as adjuvants in veterinary vaccines against foot and mouth disease, and in amplifying the protective immunity conferred by experimental vaccines against protozoal parasites such as malaria, Trypanosoma cruzi plasmodium, and the humoral response to sheep red blood cells (SRBC) (Bomford, 1982, Int. Arch. Allerg. Appl. Immun. 67:127).
The first commercially available Quillaja saponin adjuvants were crude extracts which, because of their variability, were not desirable for use in veterinary practice or in pharmaceutical compositions for man. An early attempt to purify Quillaja saponin adjuvants was made by Dalsgaard (1974, Archiv fuer die gesamte Virusforschung 44:243). Dalsgaard partially purified an aqueous extract of the saponin adjuvant material from Quillaja saponaria Molina. However, while Dalsgaard's preparation, “Quil-A,” was a definite improvement over the previously available commercial saponins, it-still exhibited considerable heterogeneity.
Subsequent analysis via high-pressure liquid chromatography showed that Quil A was in fact a heterogeneous mixture of structurally related triterpene glycosides (U.S. Pat. No. 5,057,540; Kersten et al., 1988, Infect. Immun. 56:432-438; Kensil et al., 1991, J. Immunol. 146:431-437; Kensil et al., 1991, J. Am. Vet. Med. Assoc. 199:1423-1427). However, not all of these saponins were active as adjuvants.
The four most predominant purified Quillaja saponins are QS-7,QS-17, QS-18,and QS-21 (alternatively identified as QA-7,QA-17,QA-18,and QA-21). These saponins have been purified by HPLC and low pressure silica chromatography and were found to be adjuvant active, although differing in biological activities such as hemolysis and toxicity in mice. In particular, QS-21 and QS-7 were found to be least toxic in mice (Kensil et al., 1991, J. Immunol. 146:431-437).
Due to its potent adjuvant activity and low toxicity, QS-21 (commercially available as the “Stimulon®” adjuvant) has been identified as a useful immunological adjuvant (Kensil et al., 1995, “Structural and Immunological Characterization of the Vaccine Adjuvant QS-21,” in Vaccine Design: The Subunit and Adjuvant Approach, Powell and Newman eds., Plenum Press, New York). QS-21 is a complex triterpene glycoside of quillaic acid. QS-21 is glycosylated at triterpene carbon 3,triterpene carbon 28,and carbon 5 of the second fatty acyl unit in a fatty acid domain.
More recently, QS-21 was further purified using hydrophilic interaction chromatography (HILIC) and resolved into two peaks, QS-21-V1 and QS-21-V2,which have been shown to be chemically different compounds. In C57BL/6 mice immunized with vaccines consisting of ovalbumin and either QS-21,QS-1-V1,or QS-21-V2,both of the individual components QS-21-V1 and QS-21-V2 are comparable in adjuvant effect to the original QS-21 peak (containing a mixture of 3:2 QS-21-V1 and QS-21-V2) for boosting the IgG subclasses IgG1,IgG2b, and IgG2 as well as the total IgG titer (U.S. Pat. No. 5,583,112,the entire contents of which are hereby incorporated by reference).
Quillaja saponins are structurally distinct from the saponins derived from other plant species. Two structural features that distinguish Quillaja saponaria saponins from those of other plant species are a fatty acid domain and a triterpene aldehyde at carbon 4 of the triterpene. (Kensil et al., 1995, “Structural and Immunological Characterization of the Vaccine Adjuvant QS-21,” in Vaccine Design: The Subunit and Adjuvant Approach, Powell and Newman eds., Plenum Press, New York). Modifications to the aldehyde on the triterpene indicate that this functional group may be involved in the adjuvant mechanism (Soltysik et al., 1995, Vaccine 13(15):1403-1410).
Quillaja saponins, particularly QS-7,QS-17,QS-18,and QS-21,have been found to be excellent stimulators of antibody response to soluble T-dependent protein antigens, “subunit antigens,” which are poorly immunogenic and require a potent adjuvant for maximization of immune responses. Examples of purified subunit antigens for which saponin adjuvants that augment the IgG response in mice include keyhole limpet hemocyanin (KLH), HIV-1 gp120 (Bomford et al., 1992, AIDS Res. Hum. Retroviruses 8:1765), and influenza nucleoprotein (Brett et al., 1993, Immunology 80:306). QS-7, QS-17,QS-18,and QS-21 have also been shown to stimulate potent antibody responses in mice to the antigens bovine serum albumin and cytochrome b5 (Kensil et al., 1991, J. Immunol. 146:431). The level of antibody response induced by these purified saponins was comparable to other commonly used adjuvants, e.g., complete Freund's adjuvant, and superior to aluminum hydroxide.
QS-21 has also been shown to enhance antibody responses to T-independent antigens, including unconjugated bacterial polysaccharides (White et al., 1991, “A purified saponin acts as an adjuvant for a T-independent antigen,” in: Immunobiology of Proteins and Peptides, Vol. VI (Atassi ed.), Plenum Press, New York, pp. 207-210). The immunogenicity of the vaccine was further increased by conjugating diphtheria toxoid to the polysaccharide. QS-21 enhanced the antibody response to the polysaccharide as well as the carrier, including IgG2a, IgG2b, and IgG3 responses (Coughlin et al., 1995, Vaccine 13(1):17-21).
The ability of adjuvants to modulate the isotype distribution and IgG subclass distribution of antibody response to an antigen through the promotion of Ig subclass switching has important implications for immunity to many bacterial and viral vaccines. QS-7,QS-17,QS-18,and QS-21 stimulate IgG2a response to cytochrome b5 after administration with saponin doses of 20 μg (Kensil et al., 1991, J. Immunol 146:431). In this regard, QS-21 shifts predominant IgG1 responses to a profile that includes significant IgG2b and IgG2a responses. For example, QS-21 has been shown to stimulate antigen-specific IgG2a to a number of antigens, including Borrelia burgdorferi outer surface proteins OspA and OspB (Ma et al., 1994, Vaccine 12(10):925), feline leukemia virus (FeLV) envelope gp70 (Kensil et al., 1991, J. Am. Vet. Med. Assoc. 10:1423), human cytomegalovirus (HCMV) envelope protein gB (Britt et al., 1995, J. Infect. Dis. 171:18), respiratory synctial virus (RSV) purified fusion protein (Hancock et al., 1995, Vaccine 13(4):391), and tetanus toxoid (Coughlin et al., 1995, Vaccine 13(1):17). QS-21 has also been shown to induce boostable antibody responses (Britt et al., 1995, J. Infect. Dis. 171:18-25; Helling et al., 1995, Cancer Res. 55:2783-2788).
The ability of the QS-21 adjuvant to induce class I major histocompatibility complex (MHC) antigen-restricted cytotoxic T-lymphocyte responses (CTL) after immunization with soluble proteins is a characteristic of saponin adjuvants. A number of studies have shown the ability of QS-21 to induce potent cytotoxic T-lymphocyte (CTL) responses to various antigens, including ovalbumin (Wu et al., 1994, Cell. Immunol. 154:394-406; Newman et al., 1992, J. Immunol. 148(8):2357-2362), recombinant HIV-1 gp160 protein (Wu et al., 1992, J. Immunol. 148:1519), respiratory syncytial virus (“RSV”) purified fusion protein (Hancock et al., 1995, Vaccine 13(4):391), and subunit SIVmac251 gag and env (Newman et al., 1994, AIDS Res. Hum. Retroviruses 10(7):853).
Most of the saponin adjuvant studies have been carried out in mice. However, the adjuvant activity of saponins is not limited to mice; it has also been demonstrated in humans, cats, dogs, guinea pigs, rabbits, pigs, sheep, cattle, and nonhuman primates. (Kensil et al., 1995, “Structural and Immunological Characterization of the Vaccine Adjuvant QS-21,” in Vaccine Design: The Subunit and Adjuvant Approach, Powell, M. F. and Newman, M. J. eds., Plenuim Press, New York).
Phase 1 human trials of QS-21 with GM2 ganglioside-keyhole limpet haemocyanin conjugate vaccine have been conducted in patients with malignant melanoma (Livingston et al., 1994, Vaccine 12:1275-1280.Increased immunogenicity after administration with QS-21 adjuvant was observed (Helling et al., 1995, Cancer Res. 55:2783-2788). In another set of clinical trials, QS-21 was found to be a potent immunological adjuvant that significantly increased the serological response of melanoma patients to the murine anti-idiotype antibody MELIMMUNE-1 (Livingston et al., 1995, Vaccine Res. 4(2):87).
The immune adjuvant effect of saponins is dependent upon dose. Depending upon the antigen and the species, a minimum dose level of QS-21 is required for optimum response (Kensil et al., 1991, J. Immunol. 146(2):431-7; Kensil et al., 1993, Ann N Y Acad Sci. 690:392-5; Newman et al., 1992, J. Immunol. 148(5):1519-25; Livingston et al., 1994, Vaccine 12(14):1275-80). Below this minimum dose, the immune adjuvant effect is suboptimal (either low level or absent). QS-7 also has a dose response curve (Kensil et al., 1991, J. Immunol. 146(2):431-7).
Saponins have also been discovered to elicit an innate immune response which is independent of any particular antigen. The innate immunity stimulated by saponins results in a potentiated immune system that is capable of responding to an immunological challenge in an enhanced manner. For example, saponins are capable of increasing the production of TNF-alpha, IL-6 and MIP-1-alpha in macrophage cells. In bone marrow derived dendritic cells, saponins increase the production of MIP-1-alpha and IL-1,decrease the production of I1-12 and MIP-1-beta. This effect of saponins is described in International Patent Publication No. WO 01/51083,incorporated herein in its entirety. This property of saponins is different from their adjuvant effects in that an adjuvant effect is specific to the particular antigen with which the adjuvant is administered, while the innate immunity stimulation effect results in a general enhancement of the immune system and its ability to respond to a challenge which is independent of the particular antigen used to challenge. Measurements of innate immunity, and methods of determining enhancement thereof are known in the art, and are described in International Patent Publication No. WO 01/51083.